Research Of DNA Vaccine Encoding Heterological PSCA Fusion Antigen For Anti-prostate Cancer

Objective:To construct a DNA vaccine mainly containing heterological genetic fragment encoding most cytotoxic lymphocyte epitopes of PSCA -pVAX1-PSCA₃-FcGB,and to investigate the feasibility of using PSCA as a potential target for prostate cancer therapy,and to seek for a new strategy of immutherapy of advanced and androgen-indepent prostate cancer.Methods:The heterological PSCA fusion antigen genetic fragment was synthesized by overlapping-extending-PCR.Then the former fragment was linked one by one by co-adhesive end restriction and ligation strategy until the 4 copies fragment was accomplished.The 1 to 4 copies fragments(PSCA₁-PSCA₄) were inserted into eukaryotic expression vector pCI-Fc-GPI respectively.Four kinds of corresponding plasmids were transfected into 293T cell respectively and the expression of four kinds of fusion genetic fragments were detected by immunofluorescence and flow cytometry.The PSCA₃ fragment was selected for its superior expression level in eukaryotic cells.Then the sig-PSCA₃-Fc-GPI genetic fragment was cloned into pVAX1-neo-IRES-GM/B7 vector to construct the final immunological inhanced DNA vaccine pVAX1-PSCA₃-FcGB. Immunofluorescence and flow cytometry were used to confirm the expression of PSCA₃ fragment by transfected into Cos7 cell.Finally,the anti-tumor effect of pVAX1-PSCA₃-FcGB was tested in murine prostate cancer model generated by RM-1 cell line.The animal was immunized with pVAX1-PSCA₃-FcGB DNA vaccine by intramuscular injection plus electroporation,pVAX1 and pVAX1-PSCA₁-FcGB plasmid were used as control.The inhibitory effect of tumor was investigated by observion of forming time,volume and inhibition ratio of tumor.Results:DNA sequencing conformed that the heterological PSCA fusion antigen fragment which was synchronized by overlapping-extending-PCR,was consistent to design.Enzyme digestion analysis showed that the 1 to 4 copies heterological PSCA fusion antigen fragments were constructed successfully.And corresponding eukaryotic plasmids pCI-PSCA_1/2/3/4-Fc-GPI expressed well in 293-T cells.Enzyme digestion analysis showed that the recombinant plasmid-pVAX1-PSCA₃-FcGB DNA vaccine was constructed.The expression of the DNA vaccine in Cos7 cells was demonstrated by immunofluorescence and flow cytometry.A significant delay of tumor forming time was observed in murine model with prostate cancer which immunized with pVAX1-PSCA₃-FcGB (u=-2.104,P=0.038);But inhibitory effect of tumor development was far from satisfactory.Conclusion:(1)The heterological PSCA fusion antigen genetic fragment encoding most cytotoxic lymphocyte epitopes of PSCA was synchronized by overlapping-extending-PCR.(2)A new type immunologic inhanced DNA vaccine -pVAX1-PSCA₃-FcGB,was constructed successfully.(3)This DNA vaccine can delay tumor of RM-1 cell line in vitro.